Project Summary/Abstract Studies completed since the first submission of this application show that the thioredoxin-NADP(H) inhibitor Txnip provides a mechanistic link between mitochondrial respiration and Akt-PTEN downstream signaling by controlling the reduction of disulfide bonds at the active site of PTEN and thus affects the phosphorylation state of Akt, a key regulator of insulin signaling, cell proliferation and cell survival. Signals regulating metabolism and growth are coordinately integrated via the phosphoinositide 3-kinase (PI3K)/Akt signaling pathway. The pool size of phosphatidylinositol 3,4,5-trisphosphate (PIP3), is regulated by the relative activities of PI3K and the phosphoinositide 3-phosphatase PTEN. Inactivation of a small fraction of PTEN via oxidation of critical cysteines in its active site, amplifies insulin and growth factor receptor signaling via expansion of the PIP3 pool, which activates Akt. Following PTEN's rapid reactivation via thioredoxin NADP(H)-mediated disulfide reduction, the transiently amplified PIP3 signal is attenuated by the phosphoinositide 3-phosphatase activity. Our proposed studies will examine the hypothesis that Txnip provides a proximal link between cellular redox state and Akt downstream metabolic and growth regulatory targets by maintaining NADP(H) reductive reactivation of PTEN by achieving the following specific aims: 1) To examine the hypothesis that Txnip ablation inhibits thioredoxin NADPH-dependent reactivation of PTEN by increasing cellular content NADH:NADPH ratio. We hypothesize that in non-lipogenic, oxidative tissue (skeletal muscle and hearts), due to their reduced capacity to produce NADPH, relatively small increases in NADH, caused by reduced mitochondrial respiratory electron transport, causes increases in cellular NADH: NADPH that are sufficient to inhibit the ability of thioredoxin to reduce and reactivate oxidized PTEN. 2) To elucidate the mechanism(s) by which Txnip ablation leads to impaired mitochondrial fuel oxidation 3) To examine the hypothesis that impaired mitochondrial fatty acid oxidation prevents appropriate fasting induced up-regulation of LPL gene expression in skeletal muscle and hearts of Txnip knockout mice. 4) To examine the hypothesis that impaired triglyceride lipolysis in extrahepatic tissues of Txnip knockout mice re-directs the export of hepatic fuel from VLDL triglycerides to glucose and ketone bodies. Achieving these specific aim will provide novel insights elucidating the role of Txnip in adapting fuel metabolism to nutritional state.